Thanks to its capacity to ferment sugar into ethanol and carbon dioxide, the brewer's, wine and baker's yeast Saccharomyces cerevisiae has already been used for centuries for the production of bread, wine and beer. Apart from the production of heterologous proteins, S. cerevisiae is used in biotechnology primarily in the production of ethanol for industrial purposes. In numerous industries, ethanol is used as a starting substrate for syntheses. Due to the ever decreasing oil reserves, increasing oil prices and continuously rising global need for petrol, ethanol is increasingly becoming more important as a fuel alternative.
To allow for an economic and efficient production of bioethanol, the use of lignocellulose-containing biomass, such as e.g. straw, waste material from the timber industry and agriculture and the organic proportion of everyday domestic refuse, is a prime option as a starting substrate. On the one hand, it is very cheap and, on the other hand, available in great quantities. The three major components of lignocellulose are lignin, cellulose and hemicellulose. Hemicellulose, after cellulose the second most occurring polymer, is a highly branched heteropolymer. It consists of pentoses (L-arabinose, D-xylose), uronic acids (4-0-methyl-D-glucuronic acid, D-galacturonic acid) and hexoses (D-mannose, D-galactose, L-rhamnose, D-glucose) (see FIG. 1). Even though hemicellulose can be more easily hydrolysed than cellulose, it features the pentoses L-arabinose and D-xylose, which normally cannot be converted by the yeast S. cerevisiae. 
To be able to use pentoses for fermentations, they initially have to get into the cell via the plasma membrane. Although S. cerevisiae is not able to metabolize D-xylose, it can absorb it into the cell. However, S. cerevisiae does not possess any specific transporters. The transport takes place by means of the numerous hexose transporters. However, the affinity of the transporters for D-xylose is markedly lower than that for D-glucose (Kotter and Ciriacy, 1993). In yeasts, which can metabolize D-xylose, such as e.g. P. stipitis, C. shehatae or P. tannophilus (Du Preez et al., 1986), both unspecific low-affinity transporters, which transport D-glucose and specific high-affinity proton symporters only for D-xylose are present (Hahn-Hägerdahl et al., 2001).
Utilization of D-Xylose
Different bacteria, yeasts and fungi are able to metabolize xylose. In prokaryotes and eukaryotes, the metabolization of xylose mainly differs in the type of isomerization of xylose to xylulose. In prokaryotes, the conversion of xylose to xylulose takes place by means of the enzyme xylose isomerase (XI). In eukaryotes, xylose is mostly isomerized in two steps. Initially, xylose is reduced to xylitol by the NAD(P)H-dependent xylose reductase (XR) and further converted to xylulose by the NAD-dependent xylitol dehydrogenase (XDH). The subsequent phosphorylation reaction takes place in prokaryotes and eukaryotes by means of xylulokinase (XK).
The resulting intermediate xylulose-5-phosphate is an intermediate of the pentose phosphate pathway. The major part of the xylulose-5-phosphate enters the glycolysis in the form of fructose-6-phosphate and glyceraldehyde-3-phosphate and is therein further converted to pyruvate (Schaaff-Gerstenschläger and Miosga, 1997). Under fermentative conditions, the sugar is degraded further to ethanol by the pyruvate decarboxylase and the alcohol dehydrogenase. Under aerobic conditions, pyruvate can be oxidized to carbon dioxide in the citrate cycle by means of a series of reaction steps.
Utilization of D-Xylose in S. cerevisiae 
In papers from Kötter and Ciriacy (1993), a recombinant S. cerevisiae strain, which was able to metabolize D-xylose was constructed for the first time. For this, the genes of the yeast Pichia stipitis coding for D-xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) were heterologously expressed in the yeast S. cerevisiae. In later works, the endogenous xylulokinase (XKS1) was additionally overexpressed, which improved the D-xylose absorption into the cell as well as its conversion to ethanol (Ho et al., 1998; Eliasson et al., 2000). Despite the achieved improvements, the main by-product of the xylose conversion under oxygen-limiting conditions was xylitol. This is attributed to an imbalance in the redox balance, which is caused by the reaction initially taking place in the metabolic pathway preferably using NADPH, however, the second reaction solely producing NADH (Hahn-Hägerdal et al., 2001). Under aerobic conditions, the NADH formed by the xylitol dehydrogenase can be regenerated to NAD via the respiratory chain. Under anaerobic conditions, NAD cannot be regenerated and accumulation of NADH in the cell results. Without the cofactor NAD, the xylitol dehydrogenase xylitol cannot be converted further to xylulose.
Although the xylose reductase used in the mentioned paper originates from P. stipitis, which is able to also use NADH as a cofactor, besides NADPH (Metzger and Hollenberg, 1995), the disruption of the xylose fermentation results under strict anaerobic conditions.
A solution to the problem was to introduce a redox-neutral metabolic pathway into S. cerevisiae. In prokaryotes, the conversion of xylose to xylulose takes place by means of the enzyme xylose isomerase (XI). For a complete conversion of D-xylose, only the gene XI would have to be expressed additionally as an endogenous xylulokinase is present. Although a xylose isomerase could be detected in some fungi (Tomoyeda and Horitsu, 1964; Vongsuvanglert and Tani, 1988; Banerjee et al., 1994; Rawat et al., 1996), only the xylose degradation via the enzymes xylose reductase and xylitol dehydrogenase has been shown in eukaryotes. Many efforts to heterologously express a xylose isomerase from different organisms failed (Gárdonyi and Hahn-Hägerdal, 1993). In the majority of cases, the enzymes were not functional in yeast or they were not synthesized to proteins (Sarthy et al., 1987; Amore et al., 1989; Moes of al., 1996). With high activity, only the xylose isomerase could be expressed in yeast from the obligatory anaerobic fungus Piromyces sp. E2 (Kyper et al., 2003). When heterologously overexpressing this eukaryotic xylose isomerase (Harhangi et al., 2003), S. cerevisiae was able to grow on xylose and also metabolize it under anaerobic conditions (Kuyper et al., 2003). However, further tests showed that the enzyme is strongly inhibited by xylitol, a product of the xylose conversion. Xylitol is formed unspecifically in yeast from xylose by means of aldose reductases.
U.S. Pat. No. 6,475,768 describes the use of a prokaryotic thermophilic xylose isomerase from Thermus thermophilus and variants of this, respectively, for the metabolization of xylose by yeasts. The optimal temperature for this enzyme or the variants is at a temperature (>70° C.), which is markedly higher than the temperature at which yeast grows and metabolizes (28-35° C.); however, yeast is inactive or dies off at temperatures above 40° C. However, at temperatures of about 30° C., the xylose isomerase from Thermus thermophilus and also the variants are virtually inactive. Thus, this enzyme and its variants do not permit the yeast to effectively metabolize xylose.
Therefore, a need exists in the prior art for pentose isomerases, particularly xylose isomerases allowing for an improved and more efficient pentose conversion, particularly xylose conversion.
It is thus an object of the present invention to provide improved pentose isomerases, particularly xylose isomerases, for the use in the xylose conversion, which in particular can be used for industrial yeast strains.
Xylose Isomerase (XI) Constructs and Their Use
The object is achieved according to the invention by providing a nucleic acid molecule comprising a nucleic acid sequence, which codes for a prokaryotic xylose isomerase (XI), for                the transformation of a cell, preferably for the recombinant expression and production of the xylose isomerase,        the conversion of xylose to xylulose by the cell, and/or        the formation of secondary products from xylose by the cell.        
In particular for the following uses:                the transformation of a cell, preferably for the recombinant expression/production of the prokaryotic xylose isomerase,        the conversion/metabolization, particularly fermentation, of biomaterial containing xylose,        the production of bio-based chemicals,        the production of biobutanol,        the production of bioethanol.        
“Secondary products” should be understood to mean those compounds, which the cell further produces from the xylose converted to xylulose, such as, for example, bio-based chemicals and bioalcohols.
“Bio-based chemicals” should be understood to mean chemical compounds and substances, which are obtained from biological materials and raw materials (biomass), particularly by using microorganisms.
The bio-based chemicals can be compounds, which are selected from, but not limited to: lactic acid, acetic acid, succinic acid, malic acid, 1-butanol, isobutanol, 2-butanol, other alcohols, amino acids, 1,3-propanediol, ethylene, glycerine, a β-lactam antibiotic or a cephalosporin, alkanes, terpenes, isoprenoids or the precursor molecule amorphadiene of the antimalarial drug artemisinin.
The terms “conversion” and “metabolization” are used synonymously and mean the metabolism of a substance or the conversion of a substance in the course of the metabolism, here: the conversion of xylose, particularly the conversion of xylose to xylulose, by a cell, which was transformed with a nucleic acid according to the invention. A preferred conversion/metabolization is fermentation.
The nucleic acid molecules are recombinant nucleic acid molecules. Furthermore, nucleic acid molecules according to the invention comprise dsDNA, ssDNA, PNA, CNA, RNA or mRNA or combinations thereof.
The prokaryotic xylose isomerase (XI) according to the invention comes from Clostridium phytofermentans. 
In this invention, it was achieved with a test system to express a highly functional prokaryotic xylose isomerase from Clostridium phytofermentans in the yeast S. cerevisiae. It could be shown that the xylose isomerase found allows recombinant yeasts to efficiently metabolize xylose.
The prokaryotic xylose isomerase (XI) according to the invention can be expressed in cells, particularly eukaryotic cells, in an active form. Additionally, the prokaryotic xylose isomerase (XI) according to the invention is less sensitive to an inhibition by xylitol than the eukaryotic xylose isomerase from an anaerobic fungus known from the prior art.
When the nucleic acid sequence coding for the prokaryotic xylose isomerase (XI) is expressed in a cell, the cell is imparted the capability to convert xylose to xylulose, which then may be metabolized further. Through this, the cell is able to grow on xylose as a carbon source.
The prokaryotic xylose isomerase (XI) according to the invention preferably comprises an amino acid sequence, which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical and yet more preferably 99% identical or identical to the amino acid sequence of SEQ ID NO: 1.
The nucleic acid sequence coding for a prokaryotic xylose isomerase (XI) preferably comprises a nucleic acid sequence, which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical and yet more preferably 99% identical or identical to the amino acid sequence of SEQ ID NO: 2.
The nucleic acid molecules according to the invention preferably comprise nucleic acid sequences, which are identical with the naturally occurring nucleic acid sequence or are codon-optimized for the use in a host cell.
Every amino acid is encrypted on a gene level by a codon. However, there are several different codons, which code for a single amino acid. Thus, the genetic code is degenerated. The preferred choice of a codon for a corresponding amino acid differs from organism to organism. Therefore, problems can arise in heterologously expressed genes if the host organism or the host cell has a very different codon usage. The gene can be expressed not at all or only slowly. Even in genes from different metabolic pathways within an organism, a different codon usage can be discovered. It is known that the glycolysis genes from S. cerevisiae are expressed strongly. They have a very restrictive codon usage. It can be assumed that by adapting the codon usage of the bacterial xylose isomerase gene to the codon usage of the glycolysis genes from S. cerevisiae, an improvement of the xylose conversion in yeast is achieved.
In a preferred embodiment, the nucleic acid sequence coding for a prokaryotic xylose isomerase (XI) comprises a nucleic acid sequence, which is codon-optimized for the use in a host cell.
The codon-optimization substantially preferably consists in an adaptation of the codon usage to the codon usage of the host organism/host cell, such as yeast. The codon usage of the bacterial xylose isomerase gene is preferably adapted to the codon usage of the glycolysis gene from S. cerevisiae. For further details, see also example 2 and table 1.
The nucleic acid sequence coding for a prokaryotic xylose isomerase (XI) preferably comprises a nucleic acid sequence, which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical and yet more preferably 99% identical or identical to the amino acid sequence of SEQ ID NO: 3.
The nucleic acid molecule used according to the invention is preferably a nucleic acid expression construct.
Nucleic acid expression constructs according to the invention are expression cassettes comprising a nucleic acid molecule according to the invention, or expression vectors comprising a nucleic acid molecule according to the invention or an expression cassette, for example.
A nucleic acid expression construct preferably comprises promoter and terminator sequences, the promoter being operatively linked with the nucleic acid sequence coding for a prokaryotic xylose isomerase (XI).
Preferred promoter sequences are selected from HXT7, truncated HXT7, PFK1, FBA1, PGK1, ADH1 and TDH3.
Preferred terminator sequences are selected from CYC1, FBA1, PGK1, PFK1, ADH1 and TDH3.
The nucleic acid expression construct may further comprise 5′ and/or 3′ recognition sequences and/or selection markers.
The selection marker is preferably selected from a LEU2 marker gene, a URA3 marker gene and a dominant antibiotic-resistance marker gene. A preferred dominant antibiotic-resistance marker gene is selected from genes, which impart resistances to geneticin, hygromycin and nourseothricin.
An expression vector can be selected from the group of pRS303X, p3RS305X, p3RS306X, pRS41H, pRS41K, pRS41N, pRS42H, pRS42K, pRS42N or p423HXT7-6HIS, p424HXT7-6HIS, p425HXT7-6HIS, p426HXT7-6HIS.
The cell to be transformed is preferably a eukaryotic microorganism, preferably a yeast cell or a filamentous fungal cell.
The yeast cell is preferably a member of a genus selected from the group of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Arxula and Yarrowia. 
The yeast cell is more preferably a member of a species selected from the group of S. cerevisiae, S. bulderi, S. bametti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus and K. fragilis. 
The filamentous fungal cell is preferably a member of a genus selected from the group of Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium and Penicillium. 
Xylose-Fermenting Cells
The object is achieved according to the invention by providing cells, which are transformed with a nucleic acid expression construct coding for a prokaryotic xylose isomerase (XI).
A cell according to the invention is preferably a eukaryotic cell.
A cell according to the invention, particularly a eukaryotic cell, is transformed with a nucleic acid expression construct comprising:                (a) a nucleic acid sequence coding for a prokaryotic xylose isomerase (XI),        (b) a promoter operatively linked with the nucleic acid sequence, allowing for the expression of the prokaryotic xylose isomerase (XI) in the cell.        
In this connection, the expression of the nucleic acid expression construct imparts to the cell the capability to directly isomerize xylose into xylulose.
As discussed above, the prokaryotic xylose isomerase (XI) according to the invention can be expressed in cells, particularly eukaryotic cells, in an active form such that the cells can thus directly isomerize xylose into xylulose (see also FIG. 2).
Additionally, the prokaryotic xylose isomerases (XI) according to the invention are less sensitive to an inhibition by xylitol than the eukaryotic xylose isomerases from an anaerobic fungus known from the prior art.
The inventors have introduced a redox-neutral metabolic pathway into S. cerevisiae in which the conversion of xylose to xylulose takes place by means of a xylose isomerase (XI) (FIG. 2).
When the nucleic acid sequence coding for the prokaryotic xylose isomerase (XI) is expressed in a cell, the cell is imparted the capability to convert xylose to xylulose, which then may be metabolized further. Through this, the cell is able to grow on xylose as a carbon source.
The prokaryotic xylose isomerase (XI) according to the invention preferably comes from Clostridium phytofermentans. The xylose isomerase (XI) according to the invention preferably comprises an amino acid sequence, which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical and yet more preferably 99% identical or identical to the amino acid sequence of SEQ ID NO: 1.
The promoter (b) is preferably selected from HXT7, truncated HXT7, PFK1, FBA1, PGK1, ADH1 and TDH3.
In a preferred embodiment, the nucleic acid expression construct with which a cell according to the invention is transformed is a nucleic acid molecule according to the invention, as defined herein and above.
The cell according to the invention is preferably a eukaryotic microorganism, preferably a yeast cell or a filamentous fungal cell.
A yeast cell according to the invention is preferably a member of a genus selected from the group of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Arxula and Yarrowia. 
A yeast cell according to the invention is more preferably a member of a species selected from the group of S. cerevisiae, S. bulderi, S. bametti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus and K. fragills. 
A yeast cell according to the invention is more preferably the strain Ethanol Red™ or Lallemand1.
A filamentous fungal cell according to the invention is preferably a member of a genus selected from the group of Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium and Penicillium. 
The cell according to the invention is preferably a cell maintained in a cell culture or a cultured cell.
The cells according to the invention are transiently or stably transformed with the nucleic acid expression construct or the nucleic acid molecule, as defined herein.
In one embodiment, a cell according to the invention furthermore expresses one or more enzymes, which impart to the cell the capability to produce one or more further metabolization products.
In this connection, such a further metabolization product is preferably selected from, but not limited to, the group of bio-based chemicals, such as lactic acid, acetic acid, succinic acid, malic acid, 1-butanol, isobutanol, 2-butanol, other alcohols, amino acids, 1,3-propanediol, ethylene, glycerol, a β-lactam antibiotic or a cephalosporin, alkanes, terpenes, isoprenoids or the precursor molecule amorphadiene of the antimalarial drug artemisinin.
The object is achieved according to the invention by using the cells according to the invention for the conversion/metabolization, particularly fermentation, of biomaterial containing xylose and/or for the production of bioethanol.
The object is achieved according to the invention by using the corresponding cells according to the invention for the conversion/metabolization, particularly fermentation, of biomaterial containing xylose and/or for the production of a metabolization product.
In this connection, the metabolization product is preferably selected from the group of bio-based chemicals (but not limited to this group of bio-based chemicals), such as lactic acid, acetic acid, succinic acid, malic acid, 1-butanol, isobutanol, 2-butanol, other alcohols, amino acids, 1,3-propanediol, ethylene, glycerol, a β-lactam antibiotic or a cephalosporin, alkanes, terpenes, isoprenoids or the precursor molecule amorphadiene of the antimalarial drug artemisinin.
The object is achieved according to the invention by providing a method for the production of bioethanol.
The method according to the invention comprises the following steps:                (a) converting a medium containing a xylose source with a cell according to the invention, which converts xylose to ethanol,        (b) optionally obtaining the bioethanol.        
The bioethanol is obtained by isolation, for example.
The medium may also contain another additional carbon source, particularly glucose.
The production of bioethanol preferably takes place at a rate of at least 0.03 g of ethanol per g of yeast dry weight and hour.
The ethanol yield is preferably at least 0.3 g of ethanol per g of xylose.
The object is achieved according to the invention by providing a method for the production of a metabolization product.
In this connection, such a further metabolization product is preferably selected from, but not limited to, the group of bio-based chemicals, such as lactic acid, acetic acid, succinic acid, malic acid, 1-butanol, isobutanol, 2-butanol, other alcohols, amino acids, 1,3-propanediol, ethylene, glycerol, a β-lactam antibiotic or a cephalosporin, alkanes, terpenes, isoprenoids or the precursor molecule amorphadiene of the antimalarial drug artemisinin.
The method according to the invention comprises the following steps:                (a) converting/metabolizing, particularly fermenting, a medium containing a xylose source with a corresponding cell according to the invention, which converts xylose to produce the metabolization product,        (b) optionally obtaining the metabolization product.        
The metabolization product is obtained by isolation, for example.
The medium may also contain another additional carbon source, particularly glucose.
The inventors have succeeded to introduce a redox-neutral metabolic pathway into S. cerevisiae in which the conversion of xylose to xylulose takes place by means of a xylose isomerase (XI) (FIG. 2).
In this invention, it was achieved with a test system to express a highly functional prokaryotic xylose isomerase from Clostridium phytofermentans in the yeast S. cerevisiae. It could be shown that the xylose isomerase found allows recombinant yeasts to efficiently metabolize xylose.
Furthermore, a plurality of experimental obstacles and difficulties had to be overcome in finding a functional xylose isomerase:                5 genes had to be overexpressed for the construction of the test strain MKY09.        The choice of the xylose isomerases to be tested was not trivial.        All the bacterial xylose isomerases hitherto tested showed no to very low activity in yeast.        High expenditure in the cultivation of the organisms to be tested, which were needed for the screen.        The xylose isomerase according to the invention is the first described highly active prokaryotic xylose isomerase in yeast.        The xylose isomerase according to the invention is the first xylose isomerase of cluster II (of three clusters) of xylose isomerases (see FIG. 3), which could be expressed functionally in yeasts.        The xylose isomerase according to the invention is only slightly inhibited by xylitol.        
Several reports about the difficulties with regard to the functional expression of xylose isomerases in yeast exist (Gárdonyi and Hahn-Hägerdahl, 2003; as well as reference cited therein).
The inventors have succeeded for the first time to express a prokaryotic xylose isomerase in functional form in yeasts such that they are enabled to metabolize xylose under physiological conditions and in significant quantities and to convert it to products (e.g. ethanol). As described in the prior art, this is not trivial. Numerous attempts were made and all of them were so far unsuccessful (see Sarthy et al., 1987; Amore et al., 1989; Moes et al., 1996, U.S. Pat. No. 6,475,768). The inventors have now succeeded to demonstrate that especially the C. phytofermentans xylose isomerase, in contrast to all the other, hitherto known prokaryotic enzymes, enable the yeast to metabolize xylose under physiological conditions and in significant quantities and to make products out of it.
Examples of lignocellulosic hydrolysates having a significant proportion of xylan (Hayn et al., 1993):                Grass: 16%        Wheat bran: 19%        Corn waste: 19%        
The present invention is clarified further in the following figures, sequences and examples, however, without being limited to these. The cited references are fully incorporated by reference herein. The sequences and figures show:
SEQ ID NO: 1 the protein sequence of the xylose isomerase ORF (open reading frame) of C. phytofermentans, (see also GenBank Accession Nos. ABX41597 and CP000885 (from 19 Nov. 2007)),
SEQ ID NO: 2 the nucleic acid sequence of the open reading frame (ORF) of the xylose isomerase from C. phytofermentans, (see also GenBank Accession No. CP000885 (from 19 Nov. 2007)),
SEQ ID NO: 3 the nucleic acid sequence of the open reading frame (ORF) of the xylose isomerase from C. phytofermentans in a codon-optimized form.